Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

High frequency tendon reflexes in the human soleus muscle

Tendon reflexes have been often used in studies of the human nervous system in health and disease. They have been investigated either in response to single tendon taps or to long duration vibrations. Tendon reflexes are described here in response to a high frequency vibration burst (3 cycles of a 100 Hz sine wave) applied to the Achilles tendon of standing subjects, either in quiet stance or during a forward leaning posture. The electromyogram from the soleus muscle usually showed three components separated by 10 ms which were interpreted as being three reflexes, each reflex induced by each of the three cycles in a burst. This result indicates that soleus tendon reflexes can respond in fast succession in a phasic manner when a brief high frequency vibration is applied to the Achilles tendon. This occurs in spite of possible depression of the la to motoneuron synapses and the long after hyperpolarization of the motoneurons. An interpretation of the results is that motoneurons from different subsets of the motoneuron pool respond to different cycles of the sinusoidal vibratory burst. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Comparative study of laser and LED systems of low intensity applied to tendon healing

The aim of this study was to compare the effects of Low-intensity Laser Therapy (LILT) and Light Emitting Diode Therapy (LEDT) of low intensity on the treatment of lesioned Achilles tendon of rats. The experimental model consisted of a partial mechanical lesion on the right Achilles tendon deep portion of 90 rats. One hour after the lesion, the injured animals received applications of laser/LED (685, 830/630, 880 nm), and the same procedure was repeated at 24-h intervals, for 10 days. The healing process and deposition of collagen were evaluated based on a polarization microscopy analysis of the alignment and organization of collagen bundles, through the birefringence (optical retardation-OR). The results showed a real efficiency of treatments based on LEDT and confirmed that LILT seems to be effective on healing process. Although absence of coherence of LED light, tendon healing treatment with this feature was satisfactory and can certainly replace treatments based on laser light applications. Applications of infrared laser at 830 nm and LED 880 nm were more efficient when the aim is a good organization, aggregation, and alignment of the collagen bundles on tendon healing. However, more research is needed for a safety and more efficient determination of a protocol with LED.

Muscle Transplants for Facial Reanimation Rationale and Results of Insertion Technique Using the Palmaris Longus Tendon

Thirty-one patients with unilateral long-standing facial palsy underwent I-stage reanimation with free gracilis muscle transplant innervated by the masseteric branch of the trigeminal nerve. They were divided into 2 nonrandomized groups according to insertion technique: group 1 (9 patients), interrupted suture between the free flap and the orbicularis oris of the upper and lower lip on the paralyzed side; group It (22 patients), palmaris longus tendon graft placed between the gracilis free flap and the orbicularis oris of the upper and lower lip on the nonparalyzed side. Qualitative evaluation of the smile demonstrated better results in patients from group II. Comparing the position Of the Cupid`s bow at rest, pre- and postoperatively in each patient, we observed significant improvement of facial symmetry in both groups. During smile, however, there was a significantly higher rate of centralization of the Cupid`s bow in patients submitted to reanimation with the use of the palmaris longus tendon (group II).

Spontaneous tendon rupture in systemic lupus erythematosus: association with Jaccoud`s arthropathy

Tendon rupture has rarely been described in patients with systemic lupus erythematosus. From observation of three cases of Jaccoud`s arthropathy with tendon rupture, and considering that this arthropathy is more related to an inflammatory process of the tendon sheath than to synovitis per se, the intention of this study was to review the cases of tendon rupture in patients with systemic lupus erythematosus, in the hope of determining the frequency of Jaccoud`s arthropathy associated with this complication. Systematic review using MEDLINE, Scielo and LILACS databases (1966 to 2009) and the following keywords: systemic lupus erythematosus, tendon rupture, Jaccoud`s arthropathy. Secondary references were additionally obtained. Additionally, three Brazilian systemic lupus erythematosus patients who developed tendon rupture are described. Only 40 articles obtained fulfilled the previously established criteria. They were all case reports; the number of cases reported was 52 which, together with the three cases presented herein add up to 55 cases. Forty-six patients were women aged between 19 and 71 years, with a mean age of 40.1 +/- 12.4 years, and the average duration of the disease was 10 years. The most frequently observed rupture sites were the patellar and Achilles` tendons. While almost all patients described were on various doses of corticosteroids, 16 patients concomitantly had Jaccoud`s arthropathy (29%). In conclusion, the association between Jaccoud`s arthropathy and tendon rupture in systemic lupus erythematosus has been underestimated. As almost one-third of the systemic lupus erythematosus patients with tendon rupture also have Jaccoud`s arthropathy, this arthropathy may be recognized as risk marker for tendon rupture. Lupus (2010) 19, 247-254.

Complex distal insertions of the tibialis posterior tendon: detailed anatomic and MR imaging investigation in cadavers

Purpose The purpose of this report was to demonstrate the normal complex insertional anatomy of the tibialis posterior tendon (TPT) in cadavers using magnetic resonance (MR) imaging with anatomic and histologic correlation. Material and methods Ten cadaveric ankles were used according to institutional guidelines. MR T1-weighted spin echo imaging was performed to demonstrate aspects of the complex anatomic distal insertions of the TPT in cadaveric specimens. Findings on MR imaging were correlated with those derived from anatomic and histologic study. Reults Generally, the TPT revealed a low signal in all MR images, except near the level of the medial malleolus, where the TPT suddenly changed direction and ""magic angle"" artifact could be observed. In five out of ten specimens (50%), a type I accessory navicular bone was found in the TPT. In all cases with a type I accessory navicular bone, the TPT had an altered signal in this area. Axial and coronal planes on MR imaging were the best in identifying the distal insertions of the TPT. A normal division of the TPT was observed just proximal to the insertion into the navicular bone in five specimens (100%) occurring at a maximum proximal distance from its attachment to the navicular bone of approximately 1.5 to 2 cm. In the other five specimens, in which a type I accessory navicular bone was present, the TPT directly inserted into the accessory bone and a slip less than 1.5 mm in thickness could be observed attaching to the medial aspect of the navicular bone (100%). Anatomic inspection confirmed the sites of the distal insertions of the components of the TPT. Conclusion MR imaging enabled detailed analysis of the complex distal insertions of the TPT as well as a better understanding of those features of its insertion that can simulate a lesion.

Biomechanical evaluation on tendon reinsertion by comparing trans-osseous suture and suture anchor at different stages of healing: Experimental study on rabbits

Background: Through an experimental biomechanical study on rabbits, tendon reinsertion by means of trans-osseous suture on a spongy bone bed and suture anchor were evaluated comparatively at different phases of healing. Methods: Twenty-four New Zealand White rabbits were used: 2 as pilots, 4 as the control group, and 18 as the experimental group. These 18 animals underwent sectioning and reinsertion of the Achilles tendon bilaterally, using the technique of trans-osseous suture on 1 side and suture anchor on the other. All the pelvic limbs that underwent the procedure were then immobilized for 3 weeks. The experimental group was divided into 3 groups that were sacrificed, respectively, 3, 6, and 12 weeks later. The tendon-bone complex was subjected to biomechanical tests to evaluate the parameters of maximum strength, stiffness, and yield strength. Results: There was no statistically significant difference between the suture anchor group and the trans-osseous suture group, in relation to yield strength (3 weeks, P = .222; 6 weeks, P = .465; and 12 weeks, P = .200) or maximum strength (3 weeks, P = .222; 6 weeks, P = .076; and 12 weeks, P = .078). In relation to stiffness, the suture anchor group showed a statistically significant difference only at 3 weeks of healing ( P = .032) over the trans-osseous suture group. Conclusion: The technique of suturing with an anchor was shown to be similar to the technique of trans-osseous suture for the studied parameters. Level of evidence: Basic Science Study, In-Vitro Biomechanics Study. (C) 2010 Journal of Shoulder and Elbow Surgery Board of Trustees.

Low-Intensity Pulsed Ultrasound Accelerates Healing in Rat Calcaneus Tendon Injuries

STUDY DESIGN: Controlled laboratory study. OBJECTIVE: To evaluate the effect of low-intensity therapeutic ultrasound on the murine calcaneus tendon healing process. BACKGROUND: Therapeutic ultrasound promotes formation and maturation of scar tissue. METHODS: Calcaneus tendon tenotomy and tenorrhaphy was performed on 28 Wistar rats. After the procedure, the animals were randomly divided into 2 groups. The animals in the experimental group received a 5-minute ultrasound application, once a day, at a frequency of 1 MHz, a spatial average temporal average intensity of 0.1 W/cm(2), and a spatial average intensity of 0.52 W/cm(2) at a 16-Hz frequency pulse mode (duty cycle, 20%). Data for the injured side were normalized in relation to the data from the contralateral healthy calcaneus tendon (relative values). The animals in the control group received sham treatment. After a 28-day treatment period, the animals were sacrificed and their tendons surgically removed and subjected to mechanical stress testing. The parameters analyzed were cross-sectional area (mm(2)), ultimate load (N), tensile strength (MPa), and energy absorption (mJ). RESULTS: A significant difference between groups was found for the relative values of ultimate load and tensile strength. The mean +/- SD ultimate load of the control group was -3.5% +/- 32.2% compared to 33.3% +/- 26.8% for the experimental group (P = .005). The mean tensile strength of the control group was -47.7% +/- 19.5% compared to -28.1% +/- 24.1% for the experimental group (P = .019). No significant difference was found in cross-sectional area and energy absorption. CONCLUSION: Low-intensity pulsed ultrasound produced by a conventional therapeutic ultrasound unit can positively influence the calcaneus tendon healing process in rats. J Ort hop Sports Phys Ther 2011;41(7):526-531, Epub 2 February 2011. doi:10.2519/jospt.2011.3468

Conjugated equine estrogen, raloxifene and arterial stiffness in postmenopausal women

Methods We analyzed the influence of conjugated equine estrogen (CEE) and raloxifene on arterial stiffness. Sixty-seven healthy, normotensive women 1-10 years into menopause were assigned to receive oral placebo, conjugated equine estrogen 0.625mg, or raloxifene 60mg. Arterial stiffness was evaluated by measuring the carotid-femoral and femoral-dorsalis pedis pulse wave velocity (CF PWV, FP PWV). Systolic pressure augmentation index (AI) at the carotid artery was obtained with applanation tonometry. Results Arterial stiffness was not affected by any treatment regimen: placebo (CF PWV before vs. after: 644 vs. 626 cm/s, p = 0.09; FP PWV before vs. after: 1006 vs. 1012 cm/s, p = 0.77; AI before vs. after = 30 vs. 29%, p = 0.55), CEE (CF PWV before vs. after: 642 vs. 600 cm/s, p = 0.11; FP PWV before vs. after: 952 vs. 971 cm/s, p = 0.66; AI before vs. after: 25 vs. 32%, p = 0.82), and raloxifene (CF PWV before vs. after: 636 vs. 601 cm/s, p = 0.12; FP PWV before vs. after: 964 vs. 941 cm/s, p = 0.62; AI before vs. after: 25 vs. 25%, p = 0.65). A correlation occurred between basal stiffness and the degree of reduction in indexes measured, indicating that the higher the basal stiffness, the greater the degree of reduction, particularly in the CEE group: CF PWV (r = -0.602, p = 0.001); FP PWV (r = -0.455, p = 0.022); AI (r = -0.410, p = 0.042). Conclusions Conjugated equine estrogen and raloxifene do not seem to affect arterial stiffness of healthy normotensive women less than 10 years since menopause. Reduction in arterial stiffness seems related to its basal level.

Ultrasonographic Evaluation of Achilles Tendon Repair After Percutaneous Sectioning for the Correction of Congenital Clubfoot Residual Equinus

Background: Most cases of congenital clubfoot treated with the Ponseti technique require percutaneous Achilles tenotomy to correct the residual equinus. Clinical evidence suggests that complete healing occurs between the cut tendon stumps, but there have not yet been any detailed studies investigating this reparative process. This study was performed to assess Achilles tendon repair after percutaneous section to correct the residual equinus of clubfoot treated with the Ponseti method. Method: A prospective study analyzed 37 tenotomies in 26 patients with congenital clubfoot treated with the Ponseti technique, with a minimum follow-up of 1 year after the section. The tenotomy was performed percutaneously with a large-bore needle bevel with patient sedation and local anesthesia. Ultrasonographic scanning was performed after section to ascertain that the tenotomy had been completed and to measure the stump separation. In the follow-up period, the reparative process was followed ultrasonographically and assessed at 3 weeks, 6 months, and 1 year posttenotomy. Results: The ultrasonography performed immediately after the procedure showed that in some cases, residual strands between the tendon ends persisted, and these were completely sectioned under ultrasound control. A mean retraction of 5.65 mm +/- 2.26 mm (range, 2.3 to 11.0 mm) between tendon stumps after section was observed. Unusual bleeding occurred in one case and was controlled by digital pressure, with no interference with the final treatment. After 3 weeks, ultrasonography showed tendon repair with the tendon gap filled with irregular hypoechoic tissue, and also with transmission of muscle motion to the heel. Six months after tenotomy, there was structural filling with a fibrillar aspect, mild or moderate hypoechogenicity, and tendon scar thickening when compared with a normal tendon. One year after tenotomy, ultrasound showed a fibrillar structure and echogenicity at the repair site that was similar to a normal tendon, but with persistent tendon scarring thickness. Conclusions: There is a fast reparative process after Achilles tendon percutaneous section that reestablishes continuity between stumps. The reparative tissue evolved to tendon tissue with a normal ultrasonographic appearance except for mild thickening, suggesting a predominantly intrinsic repair mechanism.

USE OF A LARGE GAUGE NEEDLE FOR PERCUTANEOUS SECTIONING OF THE ACHILLES TENDON IN CONGENITAL CLUBFOOT

Objective: To evaluate the percutaneous Achilles tendon sectioning technique using a large gauge needle for the correction of residual equinus of congenital clubfoot treated with the Ponseti method. Methods: Fifty-seven Achilles tendon sections were prospectively evaluated in thirty-nine patients with clubfoot, treated with the Ponseti method between July 2005 and December 2008. The tenotomy was performed percutaneously with a large gauge needle. Ultrasound scan was performed immediately after the section, to ascertain whether the tenotomy was complete, as well as stump separation. Results: There was complete tendon section in all cases, but the need to perform the section maneuver more than once was common, due to the persistence of the residual strands between tendon stumps. The Thompson test and dynamic ultrasound evaluation were able to detect incomplete tenotomies. The mean ultrasound measurement of the tendon gap was 5.70 +/- 2.23 mm. Two patients had abnormal bleeding, which was controlled by digital pressure and did not compromise foot perfusion. Conclusion: Percutaneous Achilles tendon sectioning with a needle proved to be efficient and safe to correct residual equinus of clubfoot treated with the Ponseti technique.

Arthrodesis of the equine proximal interphalangeal joint: a biomechanical comparison of 3-Hole 4.5 mm locking compression plate and 3-hole 4.5 mm narrow dynamic compression plate, with two transarticular 5.5 mm cortex screws

Objectives To compare the biomechanical characteristics of 2 arthrodesis techniques for the equine proximal interphalangeal joint (PIP) using either a 3-hole 4.5 mm locking compression plate (LCP) or 3-hole 4.5 mm narrow dynamic compression plate (DCP), both with 2 transarticular 5.5 mm cortex screws. Study Design Experimental. Sample Population Cadaveric adult equine forelimbs (*n=6 pairs). Methods For each forelimb pair, 1 limb was randomly assigned to 1 of 2 treatment groups and the contralateral limb by default to the other treatment group. Construct stiffness, gap formation across the PIP joint, and rotation about the PIP joint were determined for each construct before cyclic axial loading and after each of four, 5000 cycle loading regimens. After the 20,000 cycle axial loading regimen, each construct was loaded to failure. Results There were no significant differences in construct stiffness, gap formation, or sagittal plane rotation between the LCP and DCP treatment groups at any of the measured time points. Conclusion Biomechanically, fixation of the equine PIP joint with a 3-hole 4.5 mm LCP is equivalent to fixation with a 3-hole 4.5 mm narrow DCP under the test conditions used.

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of Sao Paulo, Brazil

Serum samples were collected from 582 horses from 40 stud farms in the State of Sao Paulo and tick (Acari: Ixodidae) infestations were evaluated on them. Serum samples were subjected to the complement fixation test (CFT) and a competitive inhibition ELISA (cELISA) for Babesia caballi and Theileria equi. Logistic regression analyses were performed to construct multivariate models that could explain the dependent variable (horses positive for B. caballi or T equi) as a function of the independent variables (presence or abundance of each one of the rick species found on the farms). A higher overall prevalence of B. caballi (54.1%) than of T equi (21.6%) was found by the two tests. The ticks Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) and Rbipicephalus (Boopbilus) microplus (Canestrini, 1887) were present on horses on 38 (95%), 20 (50%), and 4 (10%) farms, respectively. Infestations by D. nitens were statistically associated with B. caballi-positive horses on the farms by either the CFT or cELISA. Infestations by A. cajennense were statistically associated with T equi-positive horses on the farms by either CFT or cELISA.